The present invention relates to electrogenerated chemiluminescent methods and compositions of modified hydrocarbons and aromatic heterocyclic compounds that occur in aqueous solutions with coreactants such as tri-n-propylamine (TPrA) and peroxydisulfate (S2O82xe2x88x92) and in particular, those that provide simultaneous analyses and internal referencing. More specifically, the present invention provides substituted polycyclic organic luminescing compounds that have ring carbons optionally replaced with at least one hetero atom selected from N and S, such as substituted diphenylanthracene, thianthrene and promazine, for electrogenerated chemiluminescent applications.
Luminescing labels have been used to tag a variety of bioanalytes, such as enzymes, antibodies, antigens, peptides, nucleotides, saccharides or cells compliments of the analyte of interest, in immunoassays, DNA probe assays and fluorescent and separation assays. Of particular interest are labels which can be made to luminesce through electrochemical reaction schemes. Labels that luminesce as a result of electrochemical excitation are beneficial because of their sensitive and nonhazardous properties.
One analytical technique that exploits the benefits of these labels is electrogenerated chemiluminescence (ECL). ECL arises from an energetic electron transfer reaction between electrogenerated redox species represented by Axe2x88x92 and D+, typically radical ions, to form an excited state (A* or D*) that emits in the visible region:
A+e*xe2x86x92Axe2x88x92EAxe2x80x83xe2x80x83(1)
Dxe2x88x92e*xe2x86x92D+EDxe2x80x83xe2x80x83(2)
Axe2x88x92+D+xe2x86x92A+D*(or A*+D)xe2x80x83xe2x80x83(3)
D*xe2x86x92D+hxcexdxe2x80x83xe2x80x83(4)
Faulkner and Bard in Electroanalytical Chemistry, Vol. 10, p. 1, 1977 provide several examples of nonaqueous systems of this type.
A. W. Knight and G. M. Greenway in the Analyst, 1994, 119, 879 discuss ECL precursors that can be generated in aqueous solution even with the limited potential range imposed by the oxidation and reduction of water. ECL can be achieved by the simultaneous oxidation of tris (2,2-bipyridine)ruthenium(II) (Ru(bpy)32+) and a coreactant (XYZ) capable of generating a suitable reductant upon oxidation via an oxidative-reduction mechanism such as:
Ru(bpy)32+xe2x88x92e*xe2x86x92Ru(bpy)33+xe2x80x83xe2x80x83(5)
XYZxe2x88x92e*xe2x86x92XYZ+1xe2x80x83xe2x80x83(6)
Ru(bpy)33++XYZxe2x86x92Ru(bpy)32++XYZ+1xe2x80x83xe2x80x83(6a)
XYZ+1xe2x86x92Xx+Yy(x+y=z+1)xe2x80x83xe2x80x83(7)
Ru(bpy)33++Xxxe2x86x92Ru(bpy)32+*+Xx+1xe2x80x83xe2x80x83(8)
Ru(bpy)32+*xe2x86x92Ru(bpy)32++hxcexdxe2x80x83xe2x80x83(9)
Ru(bpy)32+ is a typical precursor (D) for these schemes and has been used in chemiluminescence (CL) reactions with amines as well as ECL investigations utilizing a number of different coreactants including oxalate (where XYz is C2O42xe2x88x92 and Xx is CO2xe2x88x92*) and aliphatic amines such as tri-n-propylamine (TPrA; where XYz is Pr3N and Xx is the radical that results from deprotonation of Pr3N+). In these ECL schemes, the coreactant can be oxidized either at the electrode via equation (6) or in solution by the emitter via equation (6a). As an alternative to the reaction scheme of equation (8), the excited state can also be generated by the sequence of equations (10) and (11), below, as previously discussed for oxalate and TPrA.
Ru(bpy)32++Xxxe2x86x92Ru(bpy)3++Xx+1xe2x80x83xe2x80x83(10)
Ru(bpy)3++Ru(bpy)33+xe2x86x92Ru(bpy)32+*+Ru(bpy)32+xe2x80x83xe2x80x83(11)
For the oxalate coreactant, the reaction scheme of equation (7) involves cleavage of the carbonxe2x80x94carbon bond while for the TPrA coreactant it is believed that this step involves the loss of a proton from the xcex1-carbon. These ECL reactions have been used for the determination of both Ru(bpy)32+ and oxalate.
To complement these oxidative-reduction examples, peroxydisulfate and Ru(bpy)32+ undergo an analogous inversion of this scheme (a reductive-oxidation mechanism, equations (12)-(14) followed by (9)) when the initial reactants are reduced, rather than oxidized, in acetonitrile (MeCN)-water solutions (1:1 volume ratio). In this case, the reduction of S2O82xe2x88x92 results in the formation of the strong oxidizing agent SO4xe2x88x92*:
Ru(bpy)32++e*xe2x86x92Ru(bpy)3+xe2x80x83xe2x80x83(12)
S2O82xe2x88x92+e*xe2x86x92SO4xe2x88x92*+SO42xe2x88x92xe2x80x83xe2x80x83(13)
Ru(bpy)3++S2O82xe2x88x92xe2x86x92Ru(bpy)32++SO4xe2x88x92*+SO4xe2x88x922xe2x88x92xe2x80x83xe2x80x83(13a)
Ru(bpy)3++SO4xe2x88x92xe2x86x92Ru(bpy)32+*+SO42xe2x88x92xe2x80x83xe2x80x83(14)
By analogy to equations (10) and (11) for oxalate and TPrA, an alternative to equation (14) for generating the excited state with peroxydisulfate is:
Ru(bpy)32++SO4xe2x88x92*xe2x86x92Ru(bpy)33++SO42xe2x88x92xe2x80x83xe2x80x83(15)
followed by equation (11) above.
The sensitivity and selectivity of these coreactant analyses has led to the recent commercial application of the Ru(bpy)32+/TPrA system. For example, electrochemiluminescent ruthenium- and osmium-containing labels have been used in methods for detecting and quantifying analytes of interest in liquid media, U.S. Pat. Nos. 5,310,687; 5,238,808; and 5,221,605, incorporated herein by reference. In addition, the application of electrogenerated chemi-luminescence (ECL) measurements to the detection of solution phase DNA intercalated with ruthenium-containing labels has been described (Carter, M. T. et al. (1990) Bioconjupate Chem 2:257-263). Although such applications provide an acceptable analysis technique, it is often necessary to provide a system that allows simultaneous analyses and internal referencing. The present invention provides additional electrochemiluminescent systems that may be used in place of or along side of existing systems.
The present invention relates to new labels where: 1) the emitter must be soluble in aqueous solution; 2) the emission wavelength must be distinct from that of Ru(bpy)32+*; 3) the oxidative or reductive electrochemistry must proceed within the relatively narrow potential range imposed by the oxidation and reduction of water; and 4) the oxidized or reduced intermediate must react with the electrogenerated coreactant intermediate allowing formation of the excited state.
An object of the present invention is to provide substituted polycyclic organic luminescing compounds, e.g., aromatic polycyclics, that may have ring carbon substitutions selected from at least one hetero N or S atom having these four (4) properties. Substituted diphenylanthracene, thianthrene and promazine exemplify such reagent systems for electrogenerated chemiluminescent applications.
Another object of the present invention is to provide substituted polycyclic organic luminescing compounds, e.g., aromatic polycyclics, that may have ring carbon substitutions, that offer a complementary label to Ru(bpy)32+ in bioanalytical applications.
It is an object of the present invention to provide label-coreactant compositions that allow for simultaneous analyses.
It is a still further object of the present invention to provide a method of detecting a plurality of analytes by (1) providing at least a first biomolecule with a luminescent label that has an emission wavelength distinct from that of Ru(bpy)32+*; (2) providing a second biomolecule analyte with a ruthenium or osmium containing label; (3) adding at least one coreactant; and (4) exposing the labelled biomolecule analytes and coreactants to electrochemical reaction or excitation and measuring the resulting luminescence to detect the various biomolecule analytes present.
A further object of the present invention is to provide a composition involving the anodic oxidation of aqueous sodium 9,10-diphenylanthracene-2-sulfonate (DPAS) in the presence of tri-n-propylamine and the cathodic reduction of DPAS in the presence of peroxydisulfate (S2O82xe2x88x92) as a coreactant in an acetonitrile (MeCN)-water solution (1:1 by volume) for ECL analysis of biomolecules (i.e., immunoassays, DNA probes). When sodium 9,10-diphenylanthracene-2 sulfonate is oxidized in the presence of TPrA or reduced with S2O82xe2x88x92, a blue ECL emission results which is characteristic of DPAS fluorescence. The spectral separation between this emission and that for Ru(bpy)32+ makes DPAS a complementary label to Ru(bpy)32+ in bioanalytical applications.
A still further object is providing 1- and 2-thianthrenecarboxylic acid (1-THCOOH and 2-THCOOH) in the presence of tri-n-propylamine (TPrA) as a coreactant in aqueous solution for electrogenerated chemiluminescence (ECL) for ECL analysis of biomolecules (i.e., immunoassays, DNA probes.
A still further object is to provide an ECL scheme of oxidizing chlorpromazine without added coreactant (TPrA) to produce an ECL emission via an unprecedented self-annihilation reaction.
These and other objects, advantages and salient features of the present invention will become more apparent from the following detailed description, non-limiting examples and annexed drawings.